Chondroitin sulfate derivative and agent for treating bladder diseases

ABSTRACT

A compound includes a group derived from chondroitin sulfate, a group derived from a steroid, and a spacer. The group derived from the chondroitin sulfate and the group derived from the steroid are covalently bonded together via the spacer, and a spacer forming molecule of the spacer is β-alanine or isoleucine. A covalent bond between the group derived from the steroid and the spacer is an ester bond, and a covalent bond between the group derived from the chondroitin sulfate and the spacer is an amide bond. The steroid is at least one selected from the group consisting of prednisolone, betamethasone, triamcinolone, triamcinolone acetonide, budesonide and fluticasone.

TECHNICAL FIELD

The present invention relates to a chondroitin sulfate derivative and anagent for treating bladder diseases that contains the chondroitinsulfate derivative.

BACKGROUND ART

Bladder diseases such as interstitial cystitis (IC) are intractablediseases presenting with symptoms such as chronic micturition andurinary urgency. However, since the cause of interstitial cystitis hasyet to be determined, the treatment thereof is dependent uponsymptomatic or empirical methods, and a definitive treatment has yet tobe established despite various attempts.

In relation thereto, chondroitin sulfate (see, for example, U.S. Pat.No. 6,083,933 or International Journal of Pharmaceutics (Ohnishi, etal., Vol. 456, pp. 113-120, 2013)) and steroids (see, for example,Guidelines for the Diagnosis and Examination of Interstitial Cystitis(Society of Interstitial Cystitis of Japan, Guideline PreparationCommittee, ed., Jan. 10, 2007) or American Urological Association (AUA)Guideline, DIAGNOSIS AND TREATMENT OF INTERSTITIAL CYSTITIS/BLADDER PAINSYNDROME (September 2014)) have been reported to be able to be used astherapeutic agents for the bladder disease of interstitial cystitis.However, although chondroitin sulfate is expected to demonstrate repaireffects on the deficient glycosaminoglycan layer, there is littleevidence of its efficacy and guidelines recommend that it not be used.In addition, steroids are associated with considerable adverse sideeffects, there is little evidence for their efficacy, and there is nobasis for their recommendation (see, for example, Guidelines for theDiagnosis and Examination of Interstitial Cystitis, Society ofinterstitial Cystitis of Japan, Guideline Preparation Committee, ed.,(Jan. 10, 2007)).

On the other hand, a compound obtained by bonding a group derived from atype of steroid in the form of prednisolone to a group derived fromchondroitin sulfate via a glycine residue (referred to as chondroitinsulfate-glycyl-prednisolone) is known to have antiarthritic activity(see, for example, International Journal of Pharmaceutics (Ohnishi, etal., Vol. 456, pp. 113-120, 2013)).

PRIOR ART LITERATURES Patent Literature

Patent Literature 1 U.S. Pat. No. 6,083,933

Non Patent Literatures

Non Patent Literature 1 Ohnishi, et al., international Journal ofPharmaceutics, Vol. 456, pp. 113-120, 2013

Non Patent Literature 2 Guidelines for the Diagnosis and Examination ofInterstitial Cystitis, Society of Interstitial Cystitis of Japan,Guideline Preparation Committee, ed., (Jan. 10, 2007)

Non Patent Literature 3 American Urological Association (AUA) Guideline,DIAGNOSIS AND TREATMENT OF INTERSTITIAL CYSTITIS/BLADDER PAIN SYNDROME(September 2014)

SUMMARY OF INVENTION Problems to be Solved by the Invention

However, International Journal of Pharmaceutics (Ohnishi, et al., Vol.456, pp. 113-1120, 2013) does not describe or suggest bladder diseasesand also does not disclose or suggest chondroitin sulfate having aspecific structure described hereinafter.

An object of the present invention is to provide a chondroitin sulfatederivative capable of reducing the systemic adverse side effectsassociated with steroids alone and demonstrating greater efficacy thanchondroitin sulfate or steroids alone, a pharmaceutical compositioncontaining that compound, and an agent for treating bladder diseasescontaining that compound.

Means for Solving the Problems

As a result of conducting extensive studies to solve the above-mentionedproblems, the inventors of the present invention discovered a compoundin which a group derived from chondroitin sulfate (CS) is covalentlybonded to a group derived from a steroid via a spacer having a specificstructure, and found that this compound demonstrates reduced systemicadverse side effects attributable to steroids and effect of moreeffectively improving bladder disease than CS or steroids alone bysustaining its pharmaceutical efficacy for a long period of time,thereby leading to completion of the present invention. In addition, theinventors of the present invention also found that the systemic adverseside effects associated with steroids can be reduced by selecting aspacer having specific properties and interposing that spacer between acovalently bonded group derived from CS and group derived from asteroid, thereby also leading to completion of the present invention.

Namely, specific means for solving the above-mentioned problems are asindicated below, and the present invention includes the followingaspects.

<1> A compound in which a group derived from CS and a group derived froma steroid are covalently bonded together via a spacer represented bygeneral formula (I):

—HN—(CH₂)_(m)—CHR¹—CO—  (I)

(in the formula, m represents an integer of 0 or 1, in the case m=0, R¹represents a group selected from a group consisting of electron-donatinggroups and steric hindrance groups, and in the case m=1, R¹ represents agroup selected from a group consisting of a hydrogen atom,electron-donating groups and steric hindrance groups).

<2> The compound described in <1>, wherein the electron-donating groupsand the steric hindrance groups represented by R¹ are groups selectedfrom a group consisting of linear alkyl groups having 1 to 12 carbonatoms, branched alkyl groups having 3 to 12 carbon atoms, cyclic alkylgroups having 3 to 12 carbon atoms, aryl groups having 6 to 10 carbonatoms, alkenyl groups having 2 to 12 carbon atoms, alkynyl groups having2 to 12 carbon atoms, and aralkyl groups having 7 to 12 carbon atoms.

<3> The compound described in <1> or <2>, wherein, in general formula(I), m=1 and R¹ represents a hydrogen atom, or m=0 and R¹ represents amethyl group, branched alkyl group having 3 to 4 carbon atoms or benzylgroup.

<4> The compound described in any of <1> to <3>, wherein, in generalformula (I), m=1 and R¹ represents a hydrogen atom, or m=0 and R¹represents a sec-butyl group.

<5>A pharmaceutical composition containing the compound described in anyof <1> to <4>.

<6> An agent for treating bladder diseases containing the compounddescribed in any of <1> to <4>,

<7> A use of the compound described in any of <1> to <4> in thetreatment of bladder diseases.

<8> A method for treating bladder diseases including administration ofthe agent for treating bladder diseases described in <6> to a subject.

<9> A urinary function ameliorant containing the compound described inany of <1> to <4>.

<10> A use of the compound described in any of <1> to <4> in theimprovement of urinary function.

<11> A method for improving urinary function including administration ofthe urinary function ameliorant described in <9> to a subject.

<12> A compound in which a group derived from CS and a group derivedfrom a steroid are covalently bonded together via a spacer, wherein thespacer exhibits a lower release rate of the steroid from the compound incomparison with the case of using a glycine residue as a spacer in thecompound.

<13> The compound described in <12>, wherein the spacer exhibits anequal or lower release rate of the steroid from the compound incomparison with the case of using a β-alanine residue as a spacer in thecompound.

<14> A method for producing a compound in which a group derived from CSand a group derived from a steroid are covalently bonded together via aspacer, including at least following steps 1 and 2.

step 1; selecting a spacer with which the release rate of the steroidfrom the compound is lower in comparison with the case of selecting aglycine residue as a spacer in the compound; and

step 2: covalently bonding the group derived from CS and the groupderived from the steroid via the spacer selected in step 1.

<15> The method described in <14>, wherein the spacer selected in step 1exhibits an equal or lower release rate of the steroid from the compoundin comparison with the case of selecting a β-alanine residue as a spacerin the compound.

Meanwhile, the above-mentioned aspects <14> and <15> can also be made tobe a method for producing a pharmaceutical composition, a method forproducing an agent for treating bladder diseases, and a method forproducing a urinary function ameliorant, which contain the compound, andthe present invention includes such aspects. In this case, a step forincorporating pharmaceutically acceptable components according to theobject of production (pharmaceutical composition, agent for treatingbladder diseases or urinary function ameliorant) can be furtherincluded.

Effects of the Invention

According to the present invention, a chondroitin sulfate derivativecapable of reducing systemic adverse side effects associated withsteroids alone and demonstrating greater efficacy than CS or steroidsalone, a pharmaceutical composition containing that compound, and anagent for treating bladder diseases containing that compound, can beprovided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph indicating the effect of CS-β-alanyl-prednisolone,CS-β-alanyl-betamethasone and CS-β-alanyl-triamcinolone acetonide onintercontraction interval in a rat hydrochloric acid-induced micturitionmodel.

FIG. 2 is a graph indicating the effect of CS-β-alanyl-triamcinoloneacetonide, CS-glycyl-triamcinolone acetonide andCS-isoleucyl-triamcinolone acetonide on intercontraction interval in arat hydrochloric acid-induced micturition model.

FIG. 3 is a graph indicating the effect of CS-β-alanyl-triamcinoloneacetonide, CS-glycyl-triamcinolone acetonide andCS-isoleucyl-triamcinolone acetonide on wet thymus weight in a rathydrochloric acid-induced micturition model.

FIG. 4 is a graph indicating the effect of CS-β-alanyl-betamethasone anda mixture of CS and betamethasone on intercontraction interval in a rathydrochloric acid-induced micturition model.

MODES FOR CARRYING OUT THE INVENTION

The term “step” in the present specification not only refers to anindependent step, but also includes a step that cannot be clearlydistinguished from another step provided the intended objective of thatstep is achieved. In addition a range of numerical values indicatedusing “to” indicates a range that includes the values described beforeand after the “to” as minimum and maximum values thereof, respectively.Moreover, unless specifically indicated otherwise, the content of eachcomponent of a composition refers to the total amount of a plurality ofthose substances present in the composition in the case a plurality oftypes of the substances are present for each component in thecomposition.

The following provides a more detailed explanation of the presentinvention using embodiments thereof.

(1) Chondroitin Sulfate Derivative

The compound according to the present invention in the form of achondroitin sulfate derivative has a group derived from CS and a groupderived from a steroid covalently bonded together via a spacerrepresented by general formula (I).

—HN—(CH₂)_(m)—CHR¹—CO—  (I)

In the formula, m represents an integer of 0 or 1. in the case to m=0,R¹ represents a group selected from a group consisting ofelectron-donating groups and steric hindrance groups, and in the casem=1, R¹ represents a group selected from a group consisting of ahydrogen atom, electron-donating groups and steric hindrance groups.

The chondroitin sulfate derivative obtained by covalently bonding agroup derived from CS and a group derived from a steroid via a spacerhaving a specific structure is able to reduce systemic adverse sideeffects associated with steroids alone and sustain an anti-inflammatoryeffect or immunosuppressive action over a long period of time. Thechondroitin sulfate derivative is able to achieve an especially superioreffect as an agent for treating bladder diseases. In the chondroitinsulfate derivative, since the spacer has a specific structure, systemicadverse side effects attributable to steroids alone can be reduced andpharmaceutical efficacy can be sustained locally, thereby enabling thedemonstration of superior effects as an agent for treating bladderdiseases in particular.

The chondroitin sulfate derivative contains at least one type of groupderived from CS. Herein, a group derived from CS refers to a groupformed by the removal of a hydroxyl group from the carboxyl group of CS.

There are no particular limitations on the CS that composes thechondroitin sulfate derivative provided it has a structure in which asulfate group is retained in an oligosaccharide skeleton formed byrepeating disaccharides consisting of D-glucuronic acid (or D-iduronicacid) and N-acetyl-D-galactosamine in disaccharide units. In addition,acidic functional groups including the carboxyl group and sulfate grouppresent in CS may be in a free state without forming a salt or may forma pharmaceutically acceptable salt.

Examples of pharmaceutically acceptable salts include salts withalkaline metal ions such as sodium salt or potassium salt, and alkalineearth metal ions such as magnesium salt or calcium salt. CS ispreferably in the form of a pharmaceutically acceptable salt with analkaline metal ion and more preferably in the form of a sodium salt fromthe viewpoints of applicability and compatibility with the living body.

The CS that composes the chondroitin sulfate derivative can be producedby a known method according to the type thereof. For example, CS can beproduced by extraction and purification from an animal-derived rawmaterial, culturing and purification from a chondroitin-producingmicroorganism and the like, oligosaccharide modification oroligosaccharide synthesis.

There are no particular limitations on the weight average molecularweight of the CS and can be suitably selected according to the objectiveand the like. The weight average molecular weight is preferably 500 to120,000, more preferably 2,500 to 60,000 and even more preferably 10,000to 40,000. Weight average molecular weight of CS can be measured bylight scattering.

The chondroitin sulfate derivative contains at least one type of groupderived from a steroid. Herein, a group derived from a steroid refers toa group formed by the removal of a hydrogen atom from a hydroxyl groupof a steroid molecule. There are no particular limitations on the sitefrom which the hydrogen atom is removed.

There are no particular limitations on the type of steroid that composesthe chondroitin sulfate derivative, and can be suitably selectedaccording to the objective and the like. Specific examples of steroidsinclude prednisolone, betamethasone, triamcinolone, triamcinoloneacetonide, budesonide and fluticasone, and at least one type selectedfrom this group is used preferably.

One type of steroid may be used alone or two or more types may be usedin combination.

The covalent bond between the group derived from a steroid and thespacer is preferably a covalent bond that can be decomposed in theliving body and is formed by a condensation reaction from the viewpointof controlling the release rate of the steroid from the chondroitinsulfate derivative, and is more preferably an ester bond. The steroid isreleased as a result of the covalent bond between the group derived froma steroid and the spacer being decomposed (and preferably bysolvolysis). The steroid may also be released by decomposing thecovalent bond between the group derived from a steroid and the spacerafter having decomposed the covalent bond between the group derived fromCS and the spacer.

In the chondroitin sulfate derivative, the group derived from CS and thespacer (—HN—(CH₂)_(m)—CHR¹—CO—) are preferably covalently bonded with anamide bond, while the group derived from a steroid and the spacer arepreferably covalently bonded with an ester bond. Namely, the compoundthat forms the spacer (hereinafter, also be referred to as thespacer-forming molecule) is preferably a compound having a structurecontaining a carboxyl group and an amino group in the molecularstructure thereof (such as an amino acid). The chondroitin sulfatederivative formed by amide bonding between a carboxyl group of CS and anamino group of the spacer-forming molecule and by ester bonding betweena hydroxyl group of the steroid and a carboxyl group of thespacer-forming molecule is preferable.

The spacer is a divalent linking group represented by general formula(I). In general formula (I), m represents an integer of 0 or 1. Here, inthe case m=0, R¹ represents an electron-donating group or a sterichindrance group. In the case m=1, R¹ represents a hydrogen atom,electron-donating group or steric hindrance group. The value of m andthe presence or absence of the electron-donating group or sterichindrance group of R¹ has an effect on the decomposition rate of theester bond between the group derived from a steroid and the spacer. Inthe case in m=0, the ester bond is more susceptible to decompositionthan in the case m=1, and the release rate of the steroid from thechondroitin sulfate derivative tends to increase.

The term “steric hindrance group” used in the present specificationrefers to a bulky group that sterically hinders a chemical reaction suchas solvolysis. In general, if R¹ of the spacer is substituted with anelectron-donating group or steric hindrance group, the covalent bondbetween the group derived from a steroid and the spacer becomes moredifficult to decompose than in the case R¹ is a hydrogen atom, therebyresulting in a decrease in the release rate of the steroid. A decreasein release rate can be suitably measured by a method describedhereinafter.

There are no particular limitations on the electron-donating group orsteric hindrance group provided it can be introduced for R¹, and can besuitably selected from commonly used electron-donating groups or sterichindrance groups. Specific examples thereof include linear alkyl groupshaving 1 to 12 carbon atoms such as a methyl group, ethyl group, propylgroup or butyl group; branched alkyl groups having 3 to 12 carbon atomssuch as an isopropyl group, isobutyl group, sec-butyl group ortert-butyl group; cyclic alkyl groups having 3 to 6 carbon atoms such asa cyclohexyl group; aryl groups having 6 to 10 carbon atoms such as aphenyl group or tolyl group; alkenyl groups having 2 to 12 carbon atomssuch as a vinyl group or allyl group; alkynyl groups having 2 to 12carbon atoms; and aralkyl groups having 7 to 12 carbon atoms such as abenzyl group. Among these groups, the electron-donating group or steric,hindrance group is preferably a group selected from a group consistingof a methyl group, branched alkyl groups having 3 to 4 carbon atoms, abenzyl group and the like. Specific examples of the spacer-formingmolecule that can be used include amino acids such as β-alanine,isoleucine, alanine, valine, leucine or phenylalanine, at least one typeof amino acid selected from this group is preferable, and at least onetype of amino acid selected from a group consisting of β-alanine,isoleucine, valine and leucine is more preferable.

In addition, other amino acids having a substituent other than thoselisted above can also be used for the above-mentioned spacer-formingmolecule. Examples of other amino acids that can be used includearginine, asparagine, serine, aspartic acid, cysteine, glutamine,glutamic acid, proline, tyrosine, tryptophan, lysine, methionine,threonine, histidine and the like.

One type of spacer-forming molecule may be used alone or two or moretypes may be used in combination.

The spacer represented by general formula (I) is preferably an aminoacid residue represented by —HN—(CH₂)₂—CO—, —HN—CH(CH(CH₃)CH₂CH₃)—CO—,—HN—CH(CH(CH₃)₂)—CO— or —HN—CHCH₂(CH(CH₃)₂)—CO—, and more preferably—HN—(CH₂)₂—CO— or —HN—CH(CH(CH₃)CH₂CH₃)—CO—, from the viewpoint ofefficacy as an agent for treating bladder diseases and inhibitingadverse side effects. In other words, the spacer-forming molecule ispreferably β-alanine, isoleucine, valine or leucine, and more preferablyβ-alanine or isoleucine.

The chondroitin sulfate derivative can adopt various structuresaccording to, for example, the structure of CS. Specific examples of thestructure of the chondroitin sulfate derivative include structurescontaining at least one type of structural unit represented by chemicalformula (II).

In formula (II), although R¹¹ respectively and independently representsa hydrogen atom or a sodium salt of a sulfate group, the cation portionof the salt of the sulfate group is not limited to the exemplifiedsodium. In addition, the number of salts of a sulfate group representedby R¹¹ is 0 to 4 and preferably 0 to 3. R¹³ represents a group selectedfrom a group consisting of steric hindrance groups and electron-donatinggroups, and R¹⁴ represents a group selected from a group consisting of ahydrogen atom, steric hindrance groups and electron-donating groups.

The chondroitin sulfate derivative may contain only one type of thestructural unit represented by the above-mentioned chemical formula, ormay contain two or more types in an arbitrary ratio and arbitrarypositional relationship. The chondroitin sulfate derivative preferablyfurther contains a structural unit normally contained in CS in additionto the structural unit represented by the above-mentioned chemicalformula. Examples of structural units normally contained in CS includestructural units in which R¹² represents ONa or OH in formula (II).However, the cation portion of the salt is not limited to theexemplified sodium.

The content ratio (weight ratio) of the steroid molecule covalentlybound with CS via the spacer to the chondroitin sulfate derivative ispreferably 3 w/w % or more, more preferably 10 w/w % or more and evenmore preferably 30 w/w % or more. Furthermore, the content ratio of thesteroid molecule can be suitably selected according to such factors asthe type of steroid molecule, its properties such as the potency of itseffect or solubility in water, and the type of spacer.

The release rate of steroid from the chondroitin sulfate derivative canbe suitably selected according to the objective and the like. Thesteroid release rate is preferably 0.1%/day to 4%/day, more preferably0.1%/day to 3%/day and even more preferably 0.5%/day to 1%/day in a 10mM phosphate buffer solution of pH 7.4 and 36° C. Here, steroid releaserate refers to the amount of change in the release ratio (%) per day,and is defined as the slope of a graph obtained by plotting time on thehorizontal axis and plotting the release ratio (%), which is the ratioof the amount of steroid released (moles) to the total number (100%) ofmoles of the group derived from a steroid contained in the chondroitinsulfate derivative, on the vertical axis.

Thus, in the case the release rate maintains a constant value during themeasurement period, the graph becomes a monotonically increasingstraight line. In this case, for example, if all of the steroidcontained in the chondroitin sulfate derivative is released in 10 daysin a 10 mM phosphate buffer solution of pH 7.4 and 36° C., the releaserate is expressed as 10%/day.

On the other hand, in the case the release rate is not constant butrather decreases over time (the graph is convex and demonstrates agentle, monotonically increasing curve), the change in the release ratioduring the initial rise period (for example, three days) is calculatedby linear approximation.

Meanwhile, the steroid released amount (amount of steroid released) ismeasured by a method suitably selected according to the type of steroid.

(2) Method for Producing Chondroitin Sulfate Derivative

The chondroitin sulfate derivative can be obtained by covalently bondinga functional group of the CS molecule (such as a carboxyl group) and afunctional group of the steroid (such as a hydroxyl group) via a spacerhaving a specific structure.

Although the following provides an explanation of an example of a methodfor producing the chondroitin sulfate derivative, the production methodthereof is not limited to the following method.

The chondroitin sulfate derivative obtained by covalently bonding agroup derived from CS and a group derived from a steroid via a spacercan be produced by a production method including, for example, the stepsindicated below:

(A) covalently bonding a hydroxyl group of a steroid by condensing witha carboxyl group of the spacer-forming molecule (ester bonding), and

(B) covalently bonding a carboxyl group of CS by condensing with anamino group of the spacer-forming molecule (amide bonding).

In step (A), a hydroxyl group of the steroid and a carboxyl group of thespacer-forming molecule are condensed to form a covalent bond. At thistime, the amino group of the spacer-forming molecule intended to reactwith CS may be protected as necessary by a commonly used method.

In step (B), a carboxyl group of CS and an amino group of thespacer-forming molecule are condensed to form a covalent bond. At thistime, the carboxyl group of the spacer-forming molecule intended toreact with the steroid may be protected as necessary by a commonly usedmethod.

The method for producing the chondroitin sulfate derivative is onlyrequired to include step (A) and step (B), and there are no limitationson the order in which these steps are carried out.

The condensation (esterification, amidation) method may be suitablyselected from commonly used methods. Examples of condensation methodsinclude a method that uses a condensing agent such as a water-solublecarbodiimide (such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride),4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloridehydrate (DMT-MM) or dicyclohexyl carbodiimide, a symmetric acidanhydride method, a mixed acid anhydride method and an active estermethod. Condensation reaction conditions are suitably selected accordingto the applied condensation reaction.

The spacer-forming molecule is preferably selected so as to obtain adesired steroid release rate. The spacer-forming molecule has a linkinggroup that links two reactive functional groups consisting of a carboxylgroup that forms an ester bond with the steroid and an amino group thatforms an amide bond with CS. The linking group is preferably selected soas to control the decomposition rate of the ester bond with the steroid.The method for selecting the spacer-forming molecule is, as previouslydescribed, selected so as to be able to achieve a desired decompositionrate by suitably combining m and R¹.

(3) Pharmaceutical Composition

The pharmaceutical composition contains at least one type of chondroitinsulfate derivative, and may further contain other components such as apharmaceutically acceptable excipient as necessary. Examples of othercomponents in addition to the pharmaceutically acceptable excipientinclude a surfactant, physiologically active substance, stabilizer,liquid medium and the like. The physiologically active substance may bethe same as or different from the steroid released from the chondroitinsulfate derivative.

There are no particular limitations on the application of thepharmaceutical composition, and is preferably used, for example, fortreating bladder diseases,

(4) Agent for Treating Bladder Diseases

The agent for treating bladder diseases is a pharmaceutical compositioncontaining at least one type of chondroitin sulfate derivative that isused to treat bladder diseases.

The term “treat” used in the present specification refers to any form oftreatment performed for a disease, and includes cure and improvement ofa disease as well as inhibition of the progression thereof (preventionof exacerbation).

Although there are no particular limitations on the form of the agentfor treating provided it is in the form of a preparation orpharmaceutical that can be administered to the urinary bladder of human,it is preferably in the form of a liquid at the time of administration,and examples thereof include solutions, suspensions and the like. Thesolution or suspension can also be administered by preparing a solutionor suspension by dissolving a powder of the chondroitin sulfatederivative at the time of use. The agent for treating bladder diseasesis preferably used for intravesical administration.

The dosage of the agent for treating bladder diseases is only requiredto be an amount sufficient for coating the entire mucous membrane insidethe urinary bladder, and in humans, for example, is about 5 mL to 200mL.

The concentration of the chondroitin sulfate derivative in the agent fortreating bladder diseases in the case of administering in the form of aliquid is only required to be that which allows passage through aurinary catheter in the case of administering using a urinary catheter,and can be suitably adjusted by a person with ordinary skill in the art.

An example of a method used to administer the agent for treating bladderdiseases consists of inserting a urinary catheter into the urinarybladder under aseptic conditions and discharging any residual urinefollowed by injecting the agent for treating bladder diseases throughthe same catheter.

After having been injected into the urinary bladder, the agent fortreating bladder diseases is thought to enable the gradual release ofsteroid over a long period of time (for example, one week or longer) asa result of the CS and steroid reaching and adhering to the bladder wallwhile remaining covalently bonded via the spacer followed by gradualdecomposition of the covalent bond between the spacer and steroid.

The agent for treating bladder diseases allows steroid to be graduallyreleased in the urinary bladder. The amount of time during which thesteroid is continued to be released in the urinary bladder (retention ofeffect) is preferably 168 hours (seven days) or more and more preferably336 hours (14 days) or more.

The intravesical administration frequency of the agent for treatingbladder diseases can be determined from the dynamics of the chondroitinsulfate derivative and the steroid released from the chondroitin sulfatederivative. Examples of the administration frequency include, but arenot limited to, once per week to once per month.

The objective of the agent for treating bladder diseases is to beapplied to the treatment of bladder diseases. The term “Bladderdiseases” used in the present specification refers to diseases and anyother abnormalities that occur in the urinary bladder, and include, forexample, acute cystitis, chronic cystitis, interstitial cystitis,hemorrhagic cystitis, radiation cystitis and overactive bladder. Theagent for treating bladder diseases is particularly preferably used forthe treatment of interstitial cystitis. The concept of interstitialcystitis may include that referred to as painful bladder syndrome (PBSor bladder pain syndrome (BPS)) and that referred to as hypersensitivebladder syndrome (HBS).

(5) Method for Treating Bladder Diseases

The method for treating bladder diseases includes a step forintravesical administration of the agent for treating bladder diseases.The method for treating bladder diseases may further include other stepsas necessary. The method for treating bladder diseases can be carriedout in the same manner as that in accordance with the explanation of“(4) Agent for Treating Bladder Diseases” of the previous section, andpreferable conditions, administration frequency and the like are thesame as previously described.

(6) Urinary Function Ameliorant

The term “urinary function ameliorant” used in the present specificationrefers to the improvement of micturition and any other abnormalitiesrelating to urinary function. Namely, the urinary function ameliorant isan agent that improves urinary function by intravesical administrationof the above-mentioned pharmaceutical composition, and includes theconcept of a micturition ameliorant. As will be understood from theexamples described hereinafter, since the above-mentioned pharmaceuticalcomposition is able to improve urinary function such as by significantlyincreasing intercontraction interval or increasing single voided volume,it can also be used to improve chronic micturition characteristic ofinterstitial cystitis. The urinary function ameliorant can beadministered in the same manner as that in accordance with theabove-mentioned explanation of the method for treating bladder diseases,and preferable conditions, administration frequency and the like are thesame as previously described.

(7) Method for Improving Urinary Function

The method for improving urinary function includes a step forintravesical administration of the urinary function ameliorant. Themethod for improving urinary function may further include other steps asnecessary. The method for improving urinary function can be carried outin the same manner as that in accordance with the explanation of “(6)Urinary Function Ameliorant” of the previous section, and preferableconditions, administration frequency and the like are the same aspreviously described.

In addition, the present invention includes the following aspects basedon the novel finding that, in a compound in which a group derived fromCS and a group derived from a steroid are covalently bonded together viaa spacer, by selecting and interposing a spacer between these groupsthat has specific properties (a spacer that lowers the release rate ofthe steroid from the compound in comparison with the case of using aspecific amino acid residue as a spacer in this compound), systemicadverse side effects attributable to the steroid can be reduced.

(8) Another Aspect of Chondroitin. Sulfate Derivative

Another aspect of the present invention of the chondroitin sulfatederivative is a compound in which a group derived from CS and a groupderived from a steroid are covalently bonded together via a spacer,wherein the spacer exhibits a lower release rate of the steroid from thecompound in comparison with the case of using a glycine residue as aspacer in the compound. In particular, a spacer is preferably employedwith which the release rate of the steroid from the above-mentionedcompound is equal to or lower than the case of using a β-alanine residueas a spacer in the above-mentioned compound.

Here, the spacer molecule in another aspect of the chondroitin sulfatederivative is only required to be a divalent linking group that linksboth the group derived from CS and the group derived from the steroid,and there are no particular limitations thereon provided the releaserate of the steroid from this compound is lower than the case ofemploying a glycine residue, and preferably a β-alanine residue, asspacer in this compound. A specific example of the spacer includes adivalent linking group represented by general formula (I) described inthe above-mentioned section (1) entitled “Chondroitin SulfateDerivative” and the like.

Here, the release rate of steroid from the above-mentioned compound canbe measured using the method described in the previously describedsection (1) entitled “Chondroitin Sulfate Derivative” or the methoddescribed in Test Example A2.

In addition, in order to select the above-mentioned spacer havingspecific properties, the spacer is determined by dissolving compoundshaving a specific structure in a 10 mM phosphate buffer solution of pH7.4 and 36° C. and comparing the values obtained by dividing the steroidrelease ratio (%) at one week later by 7 and by selecting a compoundwith which the release rate of steroid from the compound is lower thanthat of a specific amino acid residue spacer out of these compounds.

By comparing release rates determined in this manner, a spacer can beselected with which the release rate of steroid is lower in comparisonwith the case in which the spacer is a glycine residue, and preferably aβ-alanine residue.

The explanations of the CS, steroid, spacer, covalent bonds and the likethat compose another aspect of the chondroitin sulfate derivative,explanations regarding the production of another aspect of thechondroitin sulfate derivative, and the like are the same as describedin the previously described sections (1) and (3) to (7) entitled“Chondroitin Sulfate Derivative”, “Pharmaceutical Composition”, “Agentfor Treating Bladder Diseases”, “Method for Treating Bladder Diseases”,“Urinary Function Ameliorant” and “Method for Improving UrinaryFunction” provided they do not conflict with matters explained in thisother aspect of the chondroitin sulfate derivative.

(9) Another Aspect of Method for Producing Chondroitin SulfateDerivative

The present invention includes another aspect of the method forproducing a chondroitin sulfate derivative as described below. Anotheraspect of the method for producing a chondroitin sulfate derivative is amethod for producing a compound in which a group derived from CS and agroup derived from a steroid are covalently bonded together via aspacer, including (C) a step for selecting a spacer with which therelease rate of the steroid from the compound is lower in comparisonwith the case of selecting a glycine residue as a spacer in thecompound.

The production method can include a step for covalently bonding a groupderived from CS and a group derived from a steroid via the spacerselected according to that described above. There are no particularlimitations on the method used to covalently bond the above-mentionedgroups, and for example, the selected spacer and the steroid may becondensed by a known method to obtain an intermediate followed bycondensing this intermediate with CS using a known method.

For example, the steps (A) and (B) described in the previously describedsection (2) entitled “Method for Producing Chondroitin SulfateDerivative” can be included after the above-mentioned step (C). Inaddition, selection of the spacer in the above-mentioned step (C) is notnecessarily required to be carried out continuously with another step,but rather, for example, the spacer may be selected in advance followedby carrying out other subsequent steps at a later date.

Although the following provides a more detailed explanation of otheraspects of the production method, the production method is not limitedthereto.

a) A method for producing a compound in which a group derived from CSand a group derived from a steroid are covalently bonded together via aspacer, including at least following steps 1 and 2:

step 1: selecting a spacer with which the release rate of the steroidfrom the compound is lower in comparison with the case of selecting aglycine residue (and more preferably, β-alanine residue) as a spacer inthe compound; and

step 2: covalently bonding the group derived from CS and the groupderived from the steroid via the spacer selected in step 1.

b) A method for producing a compound in which a group derived from CSand a group derived from a steroid are covalently bonded together via aspacer, including at least following steps 1 to 3:

step 1: selecting a spacer with which the release rate of the steroidfrom the compound is lower in comparison with the case of selecting aglycine residue (and more preferably a β-alanine residue) as a spacer;

step 2: obtaining an intermediate by covalently bonding (such as esterbonding) the spacer selected in step 1 with the steroid; and

step 3: covalently bonding (such as amide bonding) the intermediateobtained in step 2 with CS.

In addition, the production method may also be the production method ofthe “Pharmaceutical Composition”, “Agent for Treating Bladder Diseases”and “Urinary Function Ameliorant” and the like described in thepreviously described sections (3), (4) and (6). In this case, a step foradding pharmaceutically acceptable components according to the object ofproduction (pharmaceutical composition, agent for treating bladderdiseases or urinary function ameliorant) can be further included.

The explanations of CS, steroid, spacer, covalent bonds and the like inthe production method as well as other explanations relating toproduction are the same as described in the previously described section(2) entitled “Method for Producing Chondroitin Sulfate Derivative”provided they do not conflict with matters explained in the productionmethod of the present invention.

In this manner, in a compound in which a group derived from CS and agroup derived from a steroid are covalently bonded together via aspacer, by selecting and interposing a spacer with which the releaserate of the steroid from this compound can be lowered in comparison withthe case of using a specific amino acid residue as a spacer in thiscompound, a compound, pharmaceutical composition, agent for treatingbladder diseases and/or urinary function ameliorant can be obtained inwhich systemic adverse side effects attributable to the steroid arereduced. The production method is useful for producing these products inwhich adverse side effects have been reduced.

EXAMPLES

The following provides a more detailed explanation of examples and testexamples of the present invention. However, the technical scope of thepresent invention is not limited thereby. Meanwhile, the steroid content(w/w %) of a chondroitin sulfate derivative was measured by a methoddescribed hereinafter.

Example 1 Preparation of CS Introduced with 21-β-Alanyl-Prednisolone

(1-1) Preparation of 21-(Boc-β-Alanyl)-Prednisolone

1.58 g of Boc-β-alanine (Watanabe Chemical Industries, Ltd.) weredissolved in 60 mL of dichloromethane and 20 mL of dimethylformamidefollowed by the addition of 3.00 g of prednisolone (Wako Pure ChemicalIndustries, Ltd., abbreviated as “PRED”) and 305 mg of4-dimethylaminopyridine (Wako Pure Chemical Industries, Ltd.) anddissolving therein. Subsequently, 4.00 g of water-soluble carbodiimide(Tokyo Chemical Industry Co., Ltd.) were added while cooling with icefollowed by stirring overnight at room temperature. After confirmingdisappearance of the raw material by thin layer chromatography,saturated aqueous ammonium chloride solution was added while coolingwith ice to stop the reaction. Extraction and phase separation was thencarried out using dichloromethane, toluene and water and the collectedorganic layers were sequentially washed with saturated aqueous ammoniumchloride solution, saturated aqueous sodium bicarbonate solution andsaturated aqueous sodium chloride solution. Subsequently, afterdehydrating with magnesium sulfate and filtering, the solvent wasdistilled off under reduced pressure. After dissolving the concentratewith chloroform, the solvent was again distilled off under reducedpressure. This procedure was repeated three times to obtain 4,73 g ofdesired 21-(Boc-β-alanyl)-prednisolone (Compound 1) (yield: 107%).

(1-2) Preparation of 21-β-Alanyl-Prednisolone Hydrochloride

4.73 g of Compound 1 obtained in (1-1) were dissolved in 90 mL ofdichloromethane followed by the addition of 45 mL of 4 M hydrochloricacid/ethyl acetate (Watanabe Chemical Industries, Ltd.) while coolingwith ice and stirring for 3 hours. After confirming disappearance of theraw material by thin layer chromatography, the solvent was distilled offunder reduced pressure. After washing the concentrate with diethylether, the solvent was distilled off under reduced pressure to obtain3.42 g of desired 21-β-alanyl-prednisolone hydrochloride (Compound 2)(yield: 88%).

(1-3) Preparation of CS Introduced with 21-β-Alanyl-Prednisolone

524 mg of CS (sodium salt, Seikagaku Corporation, weight averagemolecular weight: approx. 20,000, determined by light scattering) weredissolved in 10 mL of distilled water followed by the addition of 10 mLof acetone and stirring, 395 mg of Compound 2 obtained in (1-2) and 838mg of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloridehydrate (abbreviated as DMT-MM, Tokuvama Corporation) were added whilewashing with a total of 3 mL of 50% acetone/distilled water followed bystirring overnight. Subsequently, a reaction solution obtained by adding0.1 g of sodium chloride and the 50% acetone/distilled water of thewashings and dissolving the sodium chloride was sequentially added to300 mL of 90% ethanol/distilled water to form a precipitate followed bycentrifuging and discarding the supernatant. Subsequently, theprecipitate was sequentially washed with 90% ethanol/distilled water and95% ethanol/distilled water. The resulting precipitate was then driedovernight under reduced pressure to obtain 787 mg of desired CSintroduced with 21-β-alanyl-prednisolone. The content (w/w %) ofprednisolone introduced into the CS was determined to be 35 w/w % as aresult of measuring absorbance at 248 nm

Example 2 Preparation of CS Introduced with 21-β-Alanyl-Betamethasone

(2-1) Preparation of 21 -(Boc-β-Alanyl)-Betamethasone

2.0 g of Boc-β-alanine were dissolved in 50 mL of dichloromethane and 50mL of dimethylformamide followed by the addition of 4.15 g ofbetamethasone (Wako Pure Chemical Industries, Ltd., abbreviated as“BTM”). Subsequently, 389 mg of 4-dimethylaminopyridine and 2.64 g ofwater-soluble carbodiimide were added while cooling with ice followed bystirring overnight at room temperature. After confirming disappearanceof the raw material by thin layer chromatography, saturated aqueousammonium chloride solution was added to stop the reaction. Extractionand phase separation was then carried out using toluene and water andthe collected organic layers were sequentially washed with saturatedaqueous ammonium chloride solution, saturated aqueous sodium bicarbonatesolution and saturated aqueous sodium chloride solution. Subsequently,after dehydrating with magnesium sulfate and filtering, the solvent wasdistilled off under reduced pressure to obtain 5.84 g of desired21-(Boc-β-alanyl)-betamethasone (Compound 3) (yield: 98%).

(2-2) Preparation of 21-β-Alanyl-Betamethasone Hydrochloride

5.84 g of Compound 3 obtained in (2-1) were dissolved in 120 mL oftetrahydrofuran followed by the addition of 100 mL of 4 M hydrochloricacid/ethyl acetate while cooling with ice and stirring for 3 hours atroom temperature. After confirming disappearance of the raw material bythin layer chromatography, the solvent was distilled off under reducedpressure. After washing the concentrate with diethyl ether andfiltering, the solvent was distilled off under reduced pressure toobtain 4.60 g of desired 21-β-alanyl-betamethasone hydrochloride(Compound 4) (yield: 96%).

(2-3) Preparation of CS Introduced with 21-β-Alanyl-Betamethasone

10 mL of distilled water were added to 524 mg of CS (sodium salt, weightaverage molecular weight: approx. 20,000) to dissolve the CS followed bythe addition of 10 mL of acetone and stirring. 422 mg of Compound 4obtained in (2-2) and 839 mg of DMT-MM were added while washing with atotal of 3 mL of 50% acetone/distilled water followed by stirringovernight. Subsequently, a reaction solution obtained by adding 0.1 g ofsodium chloride and the 50% acetone/distilled water of the washings anddissolving the sodium chloride was sequentially added to 300 mL, of 90%ethanol/distilled water to form a precipitate followed by centrifugingand discarding the supernatant. Subsequently, the precipitate wassequentially washed with 90% ethanol/distilled water and 95%ethanol/distilled water. The resulting precipitate was then driedovernight under reduced pressure to obtain 770 mg of desired CSintroduced with 21-β-alanyl-betamethasone. The content (w/w %) ofbetamethasone introduced into the CS was determined to be 40 w/w % as aresult of measuring absorbance at 242 nm.

Example 3 Preparation of CS Introduced with 21-β-Alanyl-TriamcinoloneAcetonide

(3-1) Preparation of 21-(Boc-62 -Alanyl)-Triamcinolone Acetonide

2.63 g of Boc-β-alanine were dissolved in 40 mL of dichloromethane and10 mL, of dimethylformamide followed by the addition of 5.01 g oftriamcinolone acetonide (Wako Pure Chemical Industries, Ltd.,abbreviated as “TA”). Subsequently, 426 mg of 4-dimethylaminopyridineand 3.32 g of water-soluble carbodiimide were added while cooling withice followed by stirring overnight at room temperature. After confirmingdisappearance of the raw material by thin layer chromatography,saturated aqueous ammonium chloride solution was added to stop thereaction. Extraction and phase separation was then carried out usingtoluene and water and the collected organic layers were sequentiallywashed with saturated aqueous ammonium chloride solution, saturatedaqueous sodium bicarbonate solution and saturated aqueous sodiumchloride solution. Subsequently, after drying with magnesium sulfate andfiltering, the solvent was distilled off under reduced pressure toobtain 6.66 g of desired. 21-(Boc-β-alanyl)-triamcinolone acetonide(Compound 5) (yield.: 96%).

(3-2) Preparation of 21-β-Alanyl-Triamcinolone Acetonide Hydrochloride

6.66 g of Compound 5 obtained in (3-1) were dissolved in 90 mL oftetrahydrofuran followed by the addition of 90 mL of 4 M hydrochloricacid/ethyl acetate while cooling with ice and stirring for 3 hours atroom temperature. After confirming disappearance of the raw material bythin layer chromatography, the solvent was distilled off under reducedpressure. After washing the concentrate with diethyl ether, the solventwas distilled off under reduced pressure to obtain 5.78 g of desired21-β-alanyl-triamcinolone acetonide hydrochloride (Compound 6) (yield:97%).

(3-3) Preparation of CS Introduced with 21-β-Alanyl-TriamcinoloneAcetonide

524 mg of CS (sodium salt, weight average molecular weight: approx.20,000) were dissolved in 10 mL of distilled water followed by theaddition of 10 mL of acetone and stirring. 458 mg of Compound 6 obtainedin (3-2) and 839 mg of DMT-MM were added while washing with a total of 3mL of 50% acetone/distilled water followed by stirring overnight.Subsequently, a reaction solution obtained by adding 0.1 g of sodiumchloride and the 50% acetone/distilled water of the washings anddissolving the sodium chloride was sequentially added to 300 mL of 90%ethanol/distilled water to form a precipitate followed by centrifugingand discarding the supernatant. Subsequently, the precipitate wassequentially washed with 90% ethanol/distilled water and 95%ethanol/distilled water. The resulting precipitate was then driedovernight under reduced pressure to obtain 819 mg of desired CSintroduced with 21-β-alanyl-triamcinolone acetonide. The content (w/w %)of triamcinolone acetonide introduced into the CS was determined to be38 w/w % as a result of measuring absorbance at 242 nm.

Example 4 Preparation of CS Introduced with 21-Glycyl-TriamcinoloneAcetonide

(4-1) Preparation of 21-(Boc-Glycyl)-Triamcinolone Acetonide

1.71 g of Boc-glycine (Watanabe Chemical Industries, Ltd.) weredissolved in 30 mL of dichloromethane and 10 mL of dimethylformamidefollowed by the addition of 401 g of triamcinolone acetonide.Subsequently, 338 mg of 4-dimethylaminopyridine and 2.65 g ofwater-soluble carbodiimide were added while cooling with ice followed bystirring overnight at room temperature. After confirming disappearanceof the raw material by thin layer chromatography, saturated aqueousammonium chloride solution was added to stop the reaction. Extractionand phase separation was then carried out using toluene and water andthe collected organic layers were sequentially washed with saturatedaqueous ammonium chloride solution, saturated aqueous sodium bicarbonatesolution and saturated aqueous sodium chloride solution. Subsequently,after dehydrating with magnesium sulfate and filtering, the solvent wasdistilled off under reduced pressure to obtain 5.21 g of desired21-(Boc-glycyl)-triamcinolone acetonide (Compound 7) (yield: 96%).

(4-2) Preparation of 21-Glycyl-Triamcinolone Acetonide Hydrochloride

5.21 g of Compound 7 obtained in (4-1) were dissolved in 90 mL oftetrahydrofuran followed by the addition of 90 mL of 4 M hydrochloricacid/ethyl acetate while cooling with ice and stirring for 3 hours.After confirming disappearance of the raw material by thin layerchromatography, the solvent was distilled off under reduced pressure.After washing the concentrate with diethyl ether, the solvent wasdistilled off under reduced pressure to obtain 4.16 g of desired21-glycyl-triamcinolone acetonide hydrochloride (Compound 8) (yield:88%).

(4-3) Preparation of CS Introduced with 21-Glycyl-TriamcinoloneAcetonide 1.04 g of CS (sodium salt, weight average molecular weight:approx. 20,000) were dissolved in 20 mL of distilled water followed bythe addition of 20 mL of acetone and stirring. 806 mg of Compound 8obtained in (4-2) and 1.46 g of DMT-MM were added while washing with atotal of 20 mL of 50% acetone/distilled water followed by stirringovernight. Subsequently, a reaction solution obtained by adding 4 g ofsodium chloride and the 50% acetone/distilled water of the washings anddissolving the sodium chloride was added to 420 mL of ethanol to form aprecipitate followed by centrifuging and discarding the supernatant.Subsequently, the precipitate was sequentially washed with 80%ethanol/distilled water, 90% ethanol/distilled water, 95%ethanol/distilled water and ethanol. The resulting precipitate was thendried overnight under reduced pressure to obtain 1.34 g of desired CSintroduced with 21-glycyl-triamcinolone acetonide. The content (w/w %)of triatncinolone acetonide introduced into the CS was determined to be37 w/w % as a result of measuring absorbance at 241 nm.

Example 5 Preparation of CS Introduced with21-(Boc-Isoleucyl)-Triamcinolone Acetonide

(5-1) Preparation of 21-(Boc-Isoleucyl)-Triamcinolone Acetonide 2.24 gof Boc-isoleucine (Watanabe Chemical Industries, Ltd.) were dissolved in30 mL of dichloromethane and 10 mL of dimethylformamide followed by theaddition of 4.00 g of triamcinolone acetonide. Subsequently, 338 mg of4-dimethylaminopyridine and 2.65 g of water-soluble carbodiimide wereadded while cooling with ice followed by stirring overnight at roomtemperature. After confirming disappearance of the raw material by thinlayer chromatography, saturated aqueous ammonium chloride solution wasadded to stop the reaction. Extraction and phase separation was thencarried out using toluene and water and the collected organic layerswere sequentially washed with saturated aqueous ammonium chloridesolution, saturated aqueous sodium bicarbonate solution and saturatedaqueous sodium chloride solution. Subsequently, after dehydrating withmagnesium sulfate and filtering, the solvent was distilled off underreduced pressure to obtain 5.65 g of desired21-(Boc-isoleucyl)-triamcinolone acetonide (Compound 9) (yield: 95%),

(5-2) Preparation of 21-lsoleucyl-Triamcinolone Acetonide Hydrochloride

5.65 g of Compound 9 obtained in (5-1) were dissolved in 90 mL oftetrahydrofuran followed by the addition of 90 m of 4 M hydrochloricacid/ethyl acetate while cooling with ice and stirring for 3 hours atroom temperature. After confirming disappearance of the raw material bythin layer chromatography, the solvent was distilled off under reducedpressure. After washing the concentrate with diethyl ether, the solventwas distilled off under reduced pressure to obtain 3.05 g of desired21-isoleucyl-triamcinolone acetonide hydrochloride (Compound 10) (yield:60%).

(5-3) Preparation of CS Introduced with 21-Isoleucyl-TriamcinoloneAcetonide

1.04 g of CS (sodium salt, weight average molecular weight: approx.20,000) were dissolved in 20 mL of distilled water followed by theaddition of 20 mL of acetone and stirring. 881 mg of Compound 10obtained in (5-2) and 1.46 g of DMT-MM were added while washing with atotal of 34 mL of 50% acetone/distilled water followed by stirringovernight. Subsequently, a reaction solution obtained by adding 5 g ofsodium chloride and the 50% acetone/distilled water of the washings anddissolving the sodium chloride was sequentially added to 444 mL ofethanol to form a precipitate followed by discarding the supernatant.The precipitate was then washed with 80% ethanol/distilled waterfollowed by discarding the supernatant. 95% ethanol/distilled water wasadded followed by filtering with a filter. Precipitate remaining on thefilter was sequentially washed with 95% ethanol/distilled water andethanol and the resulting precipitate was then dried overnight underreduced pressure to obtain 1.59 g of desired CS introduced with21-isoleucyl-triamcinolone acetonide. The content (w/w %) oftriamcinolone acetonide introduced into the CS was determined to be 35w/w % as a result of measuring absorbance at 241 nm.

<Measurement of Steroid Content>

Each of the resulting compounds described above was suitably dissolvedand diluted using distilled water within the range over which absorbancedemonstrates linearity. Absorbance in the vicinity of the maximumabsorption wavelength of the measurement target was measured with aUV-visible spectrophotometer (Shimadzu Corporation).

The content w/w %) of steroid introduced into each compound wascalculated using the calculation formula indicated below.

Steroid content (w/w %)=(Abs/ε×MwST)/((WT×(100−WD)/100)/(V×Mc))×100

Abs: Absorbance

WT: Sample weight (mg)

WD: Sample weight reduction on drying (%)

ε: Molar extinction coefficient

MwST: Steroid molecular weight

V: Solution volume (mL)

Mc: Dilution factor

The molar extinction coefficients were calculated from Compounds 2, 4,6, 8 and 10 synthesized in compliance with the methods described inExamples 1 to 5. The molar extinction coefficients (ε) and molecularweights of the steroids used (MwST) of each compound are shown in Table1.

TABLE 1 Compound ε MwST 21-β-Alanyl-Prednisolone Hydrochloride 13525360.4 21-β-Alanyl-Betamethasone Hydrochloride 13402 392.521-β-Alanyl-Triamcinolone Acetonide Hydrochloride 14478 434.521-Glycyl-Triamcinolone Acetonide Hydrochloride 14730 434.521-Isoleucyl-Triamcinolone Acetonide Hydrochloride 14292 434.5

The names of the synthesized chondroitin sulfate derivatives used in thefollowing examples are abbreviated as indicated below.

TABLE 2 Compound (Chondroitin Sulfate Derivative) Abbreviation CSintroduced with 21-β-Alanyl-Prednisolone CS-βAla- PRED CS introducedwith 21-β-Alanyl-Betamethasone CS-βAla- BTM CS introduced with21-β-Alanyl-Triamcinolone Acetonide CS-βAla-TA CS introduced with21-Glycyl-Triamcinolone Acetonide CS-Gly-TA CS introduced with21-Isoleucyl-Triamcinolone Acetonide CS-Ile-TA

<Drug Release Test>

The release ratio of drug (steroid) from the chondroitin sulfatederivative was evaluated in the manner indicated below for examples usedin a drug release test.

(1) Preparation of Phosphate Buffer Solution

780 mg (5.0 mmol) of sodium dihydrogen phosphate dihydrate weredissolved in 500 mL of distilled water to prepare a Solution A. Separatetherefrom, 1.79 g (5.0 mmol) of sodium hydrogen phosphate dodecahydratewere dissolved in 500 mL of distilled water to prepare a Solution B.Solution A and Solution B were mixed at a ratio of 3:7 to prepare aphosphate buffer solution of pH 7.4.

(2) Preparation of Borate Buffer Solution

0.62 g (10 mmol) of boric acid and 0.75 g (10 mmol) of potassiumchloride were weighed out and dissolved in 500 mL of distilled water toprepare a Solution 1. Separate therefrom, a 0.1 M sodium hydroxidesolution was diluted 10-fold with distilled water to prepare a Solution2. 50 mL of Solution 1 and 7 mL of Solution 2 were added to a 100 mLvolumetric flask and brought to a volume of 100 mL with distilled waterto prepare a borate buffer solution of pH 8.0.

(3) Evaluation Method

After dissolving the chondroitin sulfate derivatives obtained in theexamples in each of the buffer solutions having respective pH values toa concentration of 5 mg/10 mL, the resulting solutions were kept for 1week in a constant temperature bath at 36° C. followed by evaluating thedrug release ratio during that time every 24 hours by high-performanceliquid chromatography (HPLC).

The drug release ratios were evaluated by measuring the ratio of theamount of drug remaining in the chondroitin sulfate derivatives and theamount of drug released using a size exclusion chromatograph (TSKgel α,Tosoh Corporation).

The conditions used during high-performance liquid chromatography aredescribed in detail below.

Analysis time: 40 min

Flow rate: 0.5 mL/min

Gradient: Isocratic

Eluent: Acetonitrile (for HPLC):physiological saline =1:2

Detector: UV detector (240 nm)

Temperature: 36° C.

Test Example A1 Change in Release Characteristics due to Difference inType of Drug

Drug release ratio (%) for a period of 1 week at 36° C. was evaluated byHPLC for CS-βAla-PRED, CS-βAla-BTM and CS-βAla-TA prepared in compliancewith the methods of Examples 1, 2 and 3. The drug release ratios wereevaluated every 24 hours. The results are shown in the table below.

TABLE 3 Elapsed CS-βAla-PRED CS-βAla-BTM CS-βAla-TA Time (days) pH 7.4pH 8.0 pH 7.4 pH 8.0 pH 7.4 pH 8.0 0 0.6% 0.6% 0.5% 0.6% 1.3% 1.3% 13.5% 7.0% 1.2% 2.9% 1.9% 2.7% 2 5.6% 12.5% 1.7% 4.8% 3.2% 4.2% 3 7.8%17.8% 2.4% 6.7% 3.7% 5.7% 4 10.0% 22.5% 3.2% 9.4% 4.5% 7.4% 5 12.0%27.3% 3.8% 11.4% 5.0% 8.6% 6 14.3% 31.6% 4.4% 13.2% 5.9% 10.0% 7 16.2%35.1% 5.0% 15.0% 6.7% 11.3% Release Rate 2.2% 4.9% 0.7% 2.1% 0.8% 1.4%(%/day)

Among the release rates for CS-βAla-PRED, CS-βAla-BTM and CS-βAla-TA,CS-βAla-PRED demonstrated the highest release rate both in the case ofpH 7.4 and pH 8.0, while the release rates for CS-βAla-BTM andCS-βAla-TA were roughly equal.

Test Example A-2 Changes in Release Characteristics Due to Difference InSpacer

Drug release ratio (%) for a period of 1 week at 36° C. was evaluated byHPLC for CS-βAla-TA, CS-Gly-TA and CS-Ile-TA prepared in compliance withthe methods of Examples 3, 4 and 5. The drug release ratios wereevaluated every 24 hours. The results are shown in the table below.

TABLE 4 Elapsed CS-βAla-TA CS-Gly-TA CS-Ile-TA Time (days) pH 7.4 pH 8.0pH 7.4 pH 8.0 pH 7.4 pH 8.0 0 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 1 0.4% 1.0%5.3% 13.1% 0.1% 0.3% 2 0.7% 2.4% 10.1% 23.2% 0.2% 0.5% 3 1.3% 3.7% 15.0%32.0% 0.3% 0.7% 4 1.8% 5.0% 19.4% 39.0% 0.4% 1.1% 5 2.3% 6.4% 23.8%44.7% 0.6% 1.4% 6 2.8% 7.8% 28.0% 49.2% 0.8% 1.8% 7 3.4% 9.0% 31.5%53.9% 0.7% 2.1% Release Rate 0.5% 1.3% 4.5% 10.6% 0.1% 0.3% (%/day)

Among the release rates for CS-βAla-TA, CS-Gly-TA and CS-Ile-TA, releaserate was the highest for CS-Gly-TA followed by CS-βAla-TA and CS-Ile-TAin that order both in the case of pH 7.4 and pH 8.0.

<In Vivo Evaluation>

Test Example B1 Evaluation of Urinary Function for CS-Steroid CompoundsUsing a Rat Hydrochloric Acid-Induced Micturition Model (SteroidComparison)

Purpose

This study was conducted for the purpose of evaluating the effect ofchondroitin sulfate derivatives having different steroids (CS-βAla-PRED,CS-βAla-BTM and CS-βAla-TA) on urinary function following single-doseadministration thereof into the bladders of hydrochloric acid-inducedmicturition model rats.

Method

Chondroitin sulfate derivatives prepared in compliance with the methodsof Examples 1, 2 and 3 were dissolved in phosphate-buffered saline toprepare the test substances indicated below.

Test Substances:

(1) Saline (control)

(2) 1.5% (w/v%) CS-βAla-PRED (FRED content: 35 w/w %)

(3) 1.5% (w/v%) CS-βAla-BTM (BTM content: 40 w/w %)

(4) 1.5% (w/v%) CS-βAla-TA (TA content: 38 w/w %)

Rats (SD strain (SPF), females, age 11 to 13 weeks) were laparotomizedunder pentobarbital general anesthesia (40 mg/kg, i.p.) followed byincising the dome of the bladder and inserting a catheter (PE50) toconstruct a bladder fistula.

Cystitis was induced using 0.4 M hydrochloric acid. The rats wereanesthetized and restrained in Bollman cages seven days afterconstruction of the bladder fistula followed by connecting a urinarycatheter, pressure transducer and syringe connected to a syringe pump inparallel. Normal intravesical pressure was measured while continuouslyinfusing physiological saline (3 mL/h). 0.4 M hydrochloric acid was thencontinuously infused into the bladder for 15 minutes (3 mL/h) to inducemicturition.

Two days after continuous instillation of hydrochloric acid, 0.2 mL ofeach test substance were administered transurethrally using a catheter(PE50) and retained in the bladder for 30 minutes under anesthesia.

Seven days after intravesical instillation of hydrochloric acid, therats were restrained in Bollman cages followed by connecting a urinarycatheter, pressure transducer and syringe connected to a syringe pump inparallel. Changes in intravesical pressure were measured whilecontinuously infusing physiological saline (3 mL/h) starting 20 minutesafter the animals emerged from anesthesia. Urinary function wasevaluated by measuring intercontraction interval and single voidedvolume after changes in intravesical pressure had stabilized.

The results are shown in FIG. 1.

Results

The results in FIG. 1 are indicated as average intercontraction interval±standard error (number of animals in each group: 3 to 7). In addition,double asterisks (**) indicate a level of significance of p<0.01 (vs.sham) according to the t-test. A double hash mark (##) indicates a levelof significance of p<0.01 (vs. control) according to Dunnett's multiplecomparison test, while a single hash mark (#) indicates a level ofsignificance of p<0.05 (vs. control) according to Dunnetts multiplecomparison test. A dollar sign ($) indicates a level of significance ofp<0.05 (vs. control) according to the t-test.

According to FIG. 1, the CS-βAla-PRED administered group. CS-βAla-BTMadministered group and CS-βAla-TA administered group each demonstratedeffect that significantly increased intercontraction interval incomparison with the control group. Similarly, with respect to singlevoided volume, the CS-βAla-PRED administered group, CS-βAla-BTMadministered group and CS-βAla-TA administered group each induced asignificant increase in single voided volume in comparison with thecontrol group.

Discussion

Since CS-βAla-PRED, CS-βAla-BTM and CS-βAla-TA clearly demonstratedeffect that improves urinary function following intravesicaladministration thereof, compounds in which CS and a steroid arecovalently bonded together via a spacer were determined to be effectiveregardless of the type of steroid.

Test Example B2 Evaluation of Urinary Function for CS-Steroid CompoundsUsing a Rat Hydrochloric Acid-Induced. Micturition Model (SpacerComparison)

Purpose

This study was conducted for the purpose of evaluating the effect ofchondroitin sulfate derivatives having different spacers (CS-βAla-TA,CS-Gly-TA and CS-Ile-TA) on urinary function following single-doseadministration thereof into the urinary bladders of hydrochloricacid-induced micturition model rats.

Method

Chondroitin sulfate derivatives prepared in compliance with the methodsof Examples 3, 4 and 5 were dissolved in phosphate-buffered saline toprepare the test substances indicated below.

Test Substances:

(1) Saline (control)

(2) 1.5% (w/v%) CS-βAla-TA (TA content: 37 w/w %)

(3) 1.5% (w/v%) CS-Gly-TA (TA content: 37 w/w %)

(4) 1.5% (w/v%) CS-Ile-TA (TA content: 35 w/w %)

Rats (SD strain (SPF), females, age 11 to 14 weeks) were laparotomizedunder anesthesia using a mixture of three anesthetic agents (Domitor,Dormicum and Vetorphale at 0.15, 2.0 and 2.5 mg/2.5 mL, respectively,and 0.5 mL/rat, i.v.) followed by incising the dome of the bladder andinserting a catheter (PE50) to construct a bladder fistula.

Cystitis was induced using 0.4 M hydrochloric acid. The rats wereanesthetized and restrained in Bollman cages seven days afterconstruction of the bladder fistula followed by connecting a urinarycatheter, pressure transducer and syringe connected to a syringe pump inparallel. Normal intravesical pressure was measured while continuouslyinfusing physiological saline (3 mL/h). 0.4 M hydrochloric acid was thencontinuously infused into the bladder for 15 minutes (3 mL/h) to inducemicturition.

Two days after continuous instillation of hydrochloric acid, 0.2 mL ofeach test substance were administered transurethrally using a catheter(PE50) and retained in the bladder for 30 minutes under anesthesia.

Seven days after intravesical instillation of hydrochloric acid, therats were restrained in Bollman cages followed by connecting a urinarycatheter, pressure transducer and syringe connected to a syringe pump inparallel. Changes in intravesical pressure were measured whilecontinuously infusing physiological saline (3 mL/h) starting 20 minutesafter the animals emerged from anesthesia. Intercontraction interval andsingle voided volume were measured after changes in intravesicalpressure had stabilized.

The results are shown in FIG. 2.

Body weight changes were determined by measuring body weight four times,namely before surgery, after surgery and immediately before theinduction with hydrochloric acid, immediately before administering thetest substance (before agent administration), and immediately beforemeasurement of intravesical pressure (cystometry), and are indicated asrelative values based on a value of 100% for the body weight prior tosurgery for each animal.

In addition, wet thymus weight was determined by collecting a sample ofthe thymus from euthanized rats following measurement of intravesicalpressure (cystometry) and measuring the weight thereof. Weight changesare shown in Table 5 while wet thymus weights are shown in FIG. 3.

TABLE 5 Before induction with Before agent Before No. of Beforehydrochloric administration cystometry Group Animals Surgery (%) acid(%) (%) (%) Sham 4 100.0 ± 0.0 99.9 ± 2.8 101.0 ± 2.2  103.8 ± 3.0 Saline 7 100.0 ± 0.0 99.8 ± 0.7 94.5 ± 1.4 99.0 ± 1.1 CS-βAla-TA 9 100.0± 0.0 100.8 ± 1.1  96.9 ± 1.3 97.8 ± 1.5 CS-Gly-TA 8 100.0 ± 0.0 101.4 ±0.7  96.7 ± 1.2 89.0 ± 1.7 CS-Ile-TA 7 100.0 ± 0.0 98.9 ± 1.3 95.7 ± 1.699.6 ± 1.2

Mean ±standard error (number of animals per group: 4 to 9)

Results

The results in FIG. 2 are indicated as average intercontraction interval±standard error (number of animals in each group: 4 to 9). In addition,a single asterisk (*) indicates a level of significance of p<0.05 (vs.sham) according to the t-test. A single hash mark (#) indicates a levelof significance of p<0.05 (vs. control) according to the t-test. Inaddition, in FIG. 3, double asterisks (**) indicate a level ofsignificance of p<0.01 (vs. control) according to the t-test.

According to FIG. 2, the CS-βAla-TA administered group, CS-Gly-TAadministered group and CS-Ile-TA administered group each demonstratedeffect that significantly increased intercontraction interval incomparison with the control group. Similarly, with respect to singlevoided volume, the CS-βAla-TA administered group, CS-Gly-TA administeredgroup and CS-Ile-TA administered group each induced a significantincrease in single voided volume in comparison with the control group.On the basis of these results, pharmacological efficacy was determinedto be demonstrated even if Ile is used for the spacer-forming molecule.CS-βAla-TA was also determined to demonstrate pharmacological efficacywith favorable reproducibility.

According to FIG. 3, significant decreases in wet thymus weight were notobserved in comparison with the control group in the CS-βAla-TAadministered group and CS-Ile-TA administered group, and significantdecreases in wet thymus weight were only observed in the CS-Gly-TAadministered group.

In addition, according to Table 5, weight loss was not observed incomparison with prior to agent administration in the CS-βAla-TAadministered group and CS-Ile-TA administered group, and prominentweight loss was only observed in the CS-Gly-TA administered group.

Discussion

CS-βAla-TA CS-Gly-TA and CS-Ile-TA clearly demonstrated effect thatimproves urinary function following intravesical administration thereof.However, adverse side effects consisting of decreased wet thymus weightand weight loss were observed for CS-Gly-TA that has a rapid drugrelease rate, and is clearly unsuitable for intravesical administration.On the other hand, systemic adverse side effects attributable tosteroids were clearly determined to be able to be reduced by controllingdrug release rate using a space-forming molecule such as βAla or Ile.

Test Example B3 Evaluation of Urinary Function for CS-Steroid CompoundsUsing a Rat Hydrochloric Acid-Induced Micturition Model (Comparison ofConjugates and Mixture)

Purpose

This study was conducted for the purpose of evaluating the effect ofchondroitin sulfate derivatives during single-dose administration ofCS-βAla-BTM and a mixture of CS and betamethasone (BTM) into the urinarybladders of hydrochloric acid-induced micturition model rats.

Method

A chondroitin sulfate derivative prepared in compliance with the methodof Example 2 was dissolved in phosphate-buffered saline to prepare thetest substances indicated below.

Test Substances:

(1) Saline (control)

(2) 3% (w/v %) CS-βAla-BTM (BTM content: 34%)

(3) Mixture of 3% CS+0.86% BTM

Rats (SD strain (SPF), females, age 10 to 13 weeks) were laparotomizedunder pentobarbital general anesthesia (40 mg/kg, i.p.) followed byincising the dome of the bladder and inserting a catheter (PE50) toconstruct a bladder fistula.

Cystitis was induced using 0.4 M hydrochloric acid. The rats wereanesthetized and restrained in Bollman cages seven days afterconstruction of the bladder fistula followed by connecting a urinarycatheter, pressure transducer and syringe connected to a syringe pump inparallel. Normal intravesical pressure was measured while continuouslyinfusing physiological saline (3 mL/h). 0.4 M hydrochloric acid was thencontinuously infused into the bladder for 15 minutes (3 mL/h) to inducemicturition.

Two days after continuous instillation of hydrochloric acid, 0.2 mL ofeach test substance were administered transurethrally using a catheter(PE50) and retained in the bladder for 30 minutes under anesthesia.

Seven days after intravesical instillation of hydrochloric acid, therats were restrained in Bollman cages followed by connecting a urinarycatheter, pressure transducer and syringe connected to a syringe pump inparallel. Changes in intravesical pressure were measured Whilecontinuously infusing physiological saline (3 mL/h) starting 20 minutesafter the animals emerged from anesthesia. Urinary function wasevaluated by measuring intercontraction interval and single voidedvolume after changes in intravesical pressure had stabilized.

The results are shown in FIG. 4. In addition, changes in rat body weightare shown in Table 6. Furthermore, changes in body weight were measuredunder the same conditions as in Test Example B2.

TABLE 6 Before induction with Before agent Before No. of Beforehydrochloric administration cystometry Group Animals Surgery (%) acid(%) (%) (%) Sham 6 100.0 ± 0.0 102.3 ± 0.6 99.6 ± 1.1 102.0 ± 1.5 Saline5 100.0 ± 0.0 103.6 ± 1.2 100.0 ± 0.7  106.1 ± 1.2 CS-βAla-BTM 8 100.0 ±0.0 102.5 ± 1.6 99.5 ± 1.3 101.7 ± 1.3 CS + BTM 5 100.0 ± 0.0 102.9 ±1.0 98.3 ± 0.6  86.8 ± 0.3 Mixture

Mean±standard error (number of animals per group: 5 to 8)

Results

The results in FIG. 4 are indicated as average intercontractioninterval:±standard error (number of animals in each group: 5 to 8). Inaddition, double asterisks (**) indicate a level of significance ofp<0.01 (vs. sham) according to the t-test. In addition, a double hashmark (##) indicates a level of significance of p<0.01 (vs. control)according to Dunnett's multiple comparison test.

According to FIG. 4, the CS-βAla-BTM administered group significantlyincreased intercontraction interval in comparison with the controlgroup. On the other hand, the CS+BTM mixture administered group did notdemonstrate effect that increases intercontraction interval. Similarly,with respect to single voided volume, although the CS-βAla-BTMadministered group induced a significant increase in single voidedvolume in comparison with the control group, the CS+BTM mixtureadministered group did not demonstrate effect that significantly inducesan increase in single voided volume.

According to Table 6, animals of the CS+BTM mixture administered groupdemonstrated prominent weight loss in comparison with the control group.On the other hand, the CS-βAla-BTM administered group was not observedto cause weight loss in comparison with the control group,

Discussion

CS-βAla-BTM clearly demonstrated effect that significantly improvesurinary function following intravesical administration thereof incomparison with a CS+BTM mixture, and was determined to clearly reducesystemic adverse side effects attributable to steroids.

The entire content of the disclosure of Japanese Patent Application No.2015-003572 (filing date: Jan. 9, 2015) is incorporated in the presentspecification by reference.

All references, patent applications and technical standards described inthe present specification are incorporated in the present specificationby reference to the same degree as in the case of the incorporation ofindividual references, patent applications and technical standards byreference being specifically and individually described.

INDUSTRIAL APPLICABILITY

The compound of the present invention is useful and can be appliedindustrially as a compound for improving bladder diseases.

1. A compound comprising: a group derived from chondroitin sulfate; agroup derived from a steroid; and a spacer wherein the group derivedfrom the chondroitin sulfate and the group derived from the steroid arecovalently bonded together via the spacer, wherein a spacer formingmolecule of the spacer is β-alanine or isoleucine, wherein a covalentbond between the group derived from the steroid and the spacer is anester bond, and a covalent bond between the group derived from thechondroitin sulfate and the spacer is an amide bond, and wherein thesteroid is at least one selected from the group consisting ofprednisolone, betamethasone, triamcinolone, triamcinolone acetonide,budesonide and fluticasone.
 2. The compound according to claim 1,wherein the chondroitin sulfate has a weight average molecular weight ofnot less than 2,500 and not more than 120,000.
 3. The compound accordingto claim I, wherein the group derived from the chondroitin sulfate formsa pharmaceutically acceptable salt.
 4. A pharmaceutical compositioncomprising the compound according to claim 1
 5. The pharmaceuticalcomposition according to claim 4, wherein the compound has a reducedsystemic adverse side effect associated with the steroid, as compared tothe steroid alone.